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. 2011 Jun 10;13(3):R59.
doi: 10.1186/bcr2896.

Epithelial to mesenchymal transition markers expressed in circulating tumour cells of early and metastatic breast cancer patients

Galatea Kallergi  1 , Maria A Papadaki, Eleni Politaki, Dimitris Mavroudis, Vassilis Georgoulias, Sophia Agelaki
Affiliations

Epithelial to mesenchymal transition markers expressed in circulating tumour cells of early and metastatic breast cancer patients

Galatea Kallergi et al. Breast Cancer Res. .

Abstract

Introduction: Epithelial to mesenchymal transition (EMT) is considered an essential process in the metastatic cascade. EMT is characterised by upregulation of vimentin, Twist, Snail, Slug and Sip1 among others. Metastasis is also associated with the presence of circulating tumour cells (CTCs) and disseminated tumour cells in the blood and bone marrow, respectively, of breast cancer patients, but the expression of EMT markers in these cells has not been reported so far.

Methods: The expression of Twist and vimentin in CTCs of 25 metastatic and 25 early breast cancer patients was investigated by using double-immunofluorescence experiments in isolated peripheral blood mononuclear cell cytospins using anti-cytokeratin (anti-CK) anti-mouse (A45-B/B3) and anti-Twist or anti-vimentin anti-rabbit antibodies.

Results: Among early breast cancer patients, vimentin-and Twist-expressing CK(+) CTCs were identified in 77% and 73% of the patients, respectively, and in 100% of the patients with metastatic breast cancer for both markers (P = 0.004 and P = 0.037, respectively). Among patients with early disease, 56% and 53% of the CK(+) CTCs were double-stained with vimentin and Twist, and the corresponding values for metastatic patients were 74% and 97%, respectively (P = 0.005 and P = 0.0001, respectively). The median expression of CK(+)vimentin(+) and CK(+)Twist(+) cells per patient in metastatic patients was 98% and 100%, and in an adjuvant chemotherapy setting the corresponding numbers were 56% and 40.6%, respectively. Triple-staining experiments revealed that all CK(+)Twist(+) or CK(+)vimentin(+) cells were also CD45(-), confirming their epithelial origin. Immunomagnetic separation of CTCs and triple-immunofluorescence with anti-CK/anti-Twist/anti-vimentin antibodies demonstrated that both mesenchymal markers could be coexpressed in the same CK(+) cell, since 64% of the total identified CTCs were triple-stained. There was a significant correlation (P = 0.005) between the number of CTCs expressing Twist and vimentin within the same setting.

Conclusions: CTCs expressing Twist and vimentin, suggestive of EMT, are identified in patients with breast cancer. The high incidence of these cells in patients with metastatic disease compared to early stage breast cancer strongly supports the notion that EMT is involved in the metastatic potential of CTCs.

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Figures

Figure 1
Figure 1
Twist and vimentin expression in HeLa cells spiked in blood of normal volunteers. ARIOL system images of HeLa cells spiked in the blood of normal volunteers. (A) Row I: Positive control for Twist. HeLa cells were stained with pan-CK A45-B/B3 antibody/secondary FITC anti-mouse antibody (green)/Twist anti-rabbit antibody/Alexa Fluor 555 anti-rabbit antibody (orange). Row II: Negative control for Twist. Cells were stained with pan-CK A45-B/B3 antibody/FITC anti-mouse (green) and Alexa Fluor 555 IgG isotype antibody. Cell nuclei were stained with DAPI (blue). Original magnification, x400. (B) Row I: Positive control for vimentin. Cells were stained with pan-CK A45-B/B3 antibody/FITC anti-mouse antibody (green)/vimentin anti-rabbit antibody/Alexa Fluor 555 anti-rabbit antibody (orange). Row II: Negative control for vimentin. Cells were stained with pan-CK A45-B/B3 antibody/FITC anti-mouse antibody (green) and Alexa Fluor 555 IgG isotype antibody. Row III: Negative control for pan-CK. Cells were stained with FITC IgG isotype antibody/vimentin anti-rabbit antibody/Alexa Fluor 555 anti-rabbit antibody (orange). Cell nuclei were stained with DAPI (blue). Original magnification, x400. ARIOL system = automated image analysis system; CK = cytokeratin; FITC = fluorescein isothiocyanate; HeLa = patient with cervical adenocarcinoma from which the cell line was derived; IgG = immunoglobulin G; DAPI = 4',6-diamidino-2-phenylindole.
Figure 2
Figure 2
Twist and vimentin expression in CTCs of early and metastatic breast cancer patients. (A) Graph I: Quantification of 25 early and 25 metastatic breast cancer patients in whom double-positive cells of each examined molecule were harvested. Graph II: Quantification of double-positive CTCs/total CTCs for each examined molecule. Graph III: Quantification of median expression per patient for each examined molecule. (B) Representative ARIOL system images of CTCs in PBMC cytospin. Row I: Cytospin double-stained with monoclonal pan-CK A45-B/B3 (green)/polyclonal Twist anti-rabbit (red) antibodies and DAPI nuclear staining. Original magnification, x400. Row II: Cytospin double-stained with monoclonal A45-B/B3 (green)/polyclonal vimentin anti-rabbit (red) antibodies and DAPI nuclear staining. Original magnification, x400. ARIOL system = automated image analysis system; CK = cytokeratin; CTCs = circulating tumour cells; PBMCs = peripheral blood mononuclear cells; DAPI = 4',6-diamidino-2-phenylindole.
Figure 3
Figure 3
Triple-immunofluorescence (CK/Twist/CD45, CK/vimentin/CD45) in CTCs. Representative ARIOL system photomicrographs of CTC cytospin after negative immunomagnetic separation in patients with metastatic breast cancer. (A) Cells were triple-stained with pan-CK A45-B/B3 antibody/Zenon Alexa Fluor 488 (green)/Twist anti-rabbit antibody/Alexa Fluor 555 anti-rabbit antibody (orange) and CD45 anti-mouse/Alexa Fluor 633 anti-mouse antibody (blue). Original magnification, x400. (B) Cells were triple-stained with pan-CK A45-B/B3 antibody/Zenon-Alexa Fluor 488 (green)/vimentin anti-rabbit antibody/Alexa Fluor 555 anti-rabbit antibody (orange) and CD45 anti-mouse/Alexa Fluor 633 anti-mouse antibody (blue). Original magnification, x400. ARIOL system = automated image analysis system; CK = cytokeratin; CTCs = circulating tumour cells.
Figure 4
Figure 4
Coexpression of CK, Twist and vimentin in the same cell. Representative confocal laser-scanning photomicrographs of CTC cytospin after negative immunomagnetic separation in a patient with metastatic breast cancer. Cells were triple-stained with pan-CK A45-B/B3 antibody/Zenon Alexa Fluor 488 (green)/Twist anti-mouse/Alexa Fluor 633 anti-mouse antibody/vimentin anti-rabbit/Alexa Fluor 555 anti-rabbit antibody (orange). Original magnification, x600. (A) A CTC expressing CK, Twist and vimentin. (B) A CTC expressing CK and Twist, but not vimentin. CK = cytokeratin; CTCs = circulating tumour cells.
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